Morphological and molecular characterization of Fusarium spp., the fungi associated with mulberry root rot disease in north-eastern Thailand

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1 THAI JOURNAL OF BOTANY 2(1): (1): Morphological and molecular characterization of Fusarium spp., the fungi associated with mulberry root rot disease in north-eastern Thailand WARAPORN SUTTHISA 1, NIWAT SANOAMUANG 1,2* AND SARIDIPORN CHUPRAYOON 3 1 Plant Pathology Division, Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand 2 Applied Taxonomic Research Center, Khon Kaen University, Khon Kaen 40002, Thailand 3 The Sirikit Institute of Sericulture, Ministry of Agriculture and Cooperative, Bangkok, Thailand ABSTRACT. This study was conducted to isolate and identify Fusarium spp. the associated causal agents of mulberry root rot disease. One hundred and fty-seven isolates of Fusarium spp. were obtained from 5 mulberry plantations during 2007 to All species were identi ed morphologically with particular emphasis on macroconidia, microconidia, the size and types of conidiophores, and the presence or absence of chlamydospores. The species associated with mulberry roots and surrounding rhizosphere were identi ed to 11 species including 102 isolates of Fusarium solani, 26 of F. moniliforme, 16 of F. oxysporum, 3 each of F. phaseoli and F. dlamini, 2 of F. culmorum, 1 each of F. dimerum, F. graminearum, F. beomiforme, F. scirpi and F. anthophilum. Genetic variation of 30 isolates of Fusarium spp. was studied using random ampli ed polymorphic DNA (RAPD) markers. Four primers A02, A03, A07 and A09 produced ngerprint pro les, which clearly distinguished between the different species of Fusarium spp. The high level of genetic variability among species of Fusarium allowed ngerprinting of most of the isolates by examining patterns produced by several primers. The results suggest that RAPD-PCR is a useful method for analyzing genetic variation within and between Fusarium spp. Keywords: Fusarium spp., mulberry root rot, RAPD * Corresponding author: niwat@kku.ac.th Received: 23 November 2009 Accepted: 31 March 2010

2 26 Waraporn Sutthisa et al. INTRODUCTION Root rot disease is considered to be the most destructive disease of mulberry (Morus alba L.) in the northeast of Thailand. The disease can be found wherever the plant is grown and direct losses on the crop due to the disease can reach 100% (Sanoamuang & Saksirirat, 1984). The indirect loss on the sericulture production is very large and therefore the economic importance of these diseases should never be underestimated. The disease was rst reported in Since then its etiology has been extensively studied (Sanoamuang & Saksirirat, 1984; Winidsanan, 1987; Chancharean, 1990; Yamakawa et al., 1991; Chuprayoon et al., 1994; Thaworn-anukulkid, 1996; Saksung et al., 1997; Kaosiri, 1998). Symptoms of the disease appear as sudden leaf withering starting from the bottom of the branch upwards, followed by defoliation in isolated patches in the plantation. The bark of the roots peels off easily with a particular odour and the plants suddenly die. Through irrigation water, soil and garden implements, the disease spreads quickly to nearby mulberry trees both in the same row and the adjacent rows where the root zones are in overlapping proximity (Sanoamuang & Saksirirat, 1984; The Hindu, 2004). Sanoamuang et al. (1986) reported that Fusarium solani f.sp. mori was always found associated with the mulberry roots in both healthy and diseased trees collected from all sericulture experiment stations in the northeastern of Thailand. The genus Fusarium has a worldwide distribution. Its different species are considered to be some of the most important plant disease pathogens (Nelson et al., 1983). Summerell et al. (2003) reported that a utilitarian approach to Fusarium identification including morphological, biological and phylogenetical species concepts. In general, morphological species concepts are based on the similarity of observable morphological characters, e.g. spore size and shape. Biological species concepts required that members of the same species are sexually cross-fertile and that the progeny of the crosses are both viable and fertile. Finally, DNA sequences have been used to generate characters that usually are treated cladistically to form phylogenies. A molecular approach using random amplified polymorphic DNA (RAPD) markers as a diagnostic tool for the identi cation of Fusarium spp. has been reported (Achenbach et al., 1996). RAPD analysis has many advantages as a mean of characterizing genetic variability such as speed, low cost, minimal requirement for DNA, and lack of radioactivity. Major polymorphisms

3 Morphological and molecular characterization o Fusarium spp. 27 in RAPD pattern indicate genetic distinctness and can be used to distinguish unrelated groups. Minor polymorphisms may indicate genetic distinctness within groups or may occur because of experimental variability and therefore, must be veri ed by repetition (Nelson et al., 1997). The purpose of this study was to isolate and identify Fusarium spp., the causal agent associated with mulberry root rot disease in northeastern Thailand using morphological characteristics and RAPD pattern analysis. MATERIALS AND METHODS Isolation of fungi Root and soil around the rhizosphere of mulberry trees exhibiting or not exhibiting symptoms of root rot were used to isolate associated pathogens. Mulberry roots and soil were collected at elds in ve different areas of northeastern Thailand in June 2007 to February 2008: Silk innovation center (Mahasarakham University, 7 June 2007), Ban Kwao-Yai (Kantarawichai District, Mahasarakham Province, 24 June 2007), Ban Kee (Cheangyuen District, Mahasarakham Province, 14 July 2007), Ban Wan-fai (Art-Sahmaht District, Roi-et Province, 6 October 2007) and Khon-Kaen University (1 February 2008). The roots were cut from 5 infected and 5 non-infected plants and the root tissues were washed in running tap water, cut into pieces approximately 3 to 5 mm long. They were surface disinfected in 0.6% sodium hypochlorite for 5 min, rinsed twice in sterile distilled water, blotted dry on sterile lter paper, and then plated on potato dextrose agar (PDA, Merck Laboratories, Germany) added 100 mg/l rifampicin in 9 cm diameter Petri dishes. The dishes with tiny pieces of mulberry root were incubated at 28 ºC and they were examined periodically after 3-5 days. Emerging fungal mycelia were transferred onto new PDA plates. Soil samples were collected from the stem base to a depth of 15 cm 3 points around the plant (100 g per plant) and 5 plants per infected and non-infected eld. In order to assay the organisms in the soil, each sample from infected eld or noninfected elds were thoroughly mixed and 10 g of each mixed sample was transferred to 90 ml of de-ionized distilled water in a 250 ml Erlenmeyer ask. The asks were agitated on a rotary shaker for 30 min and 10-fold dilutions were made serially from the original suspension. From each of the dilution, 100 l aliquot was spread over the surface of rose bengal agar in a 9 cm diameter Petri dish. There were 3 plates per dilution, and all plates were incubated at 28 ºC for 3 days.

4 28 Waraporn Sutthisa et al. Emerging colonies were cut and transferred individually onto new PDA plates. Identi cation of isolates All single spore cultures of Fusarium isolates were identi ed after Booth (1977), Nelson et al. (1994), Seifert (1996) and Aoki et al. (2003). After the isolates were grown on PDA for 10 days, colony growth and spore morphology and size were characterized. Slide culture technique was used to observe detailed morphological structures of taxonomic value based on the form of macroconidia and microconidia produced on monophialides or polyphialides (Rodrigues & Menezes, 2005). All single spore isolation cultures were freeze-dried and kept in a refrigerator. Genomic DNA extraction Isolates were grown in potato dextrose broth (PDB) for 7 days at 28 ºC on a shaker at 120 rpm. Mycelia were harvested by ltration through lter paper (Whatman No.1) and freeze-dried for 24 hr. Total DNA extraction was prepared by a modified method of Zang et al. (2005). Approximately 50 mg of freezedried mycelium was grounded in liquid nitrogen. The resulting powder was suspended in 0.7 ml of lysis buffer (400 mm Tris-HCl (ph 8.0), 60mM EDTA (ph 8.0), 150 mm NaCl, 1% SDS) and 3 l of 2-mercaptoethanol. Samples were incubated at 65 ºC for 30 min and 150 l of potassium acetate (ph 4.8) was added and mixed gently. This mixture was centrifuged for 5 min at 12,000 rpm. The aqueous phase was collected, extracted with an equal volume of chloroform : isoamyl alcohol (24:1) mixed gently and centrifuged for 5 min at 12,000 rpm. The aqueous phase was collected and DNA was then precipitated by adding with an equal volume of cold isopropanol, incubated at -20 ºC for 30 min and pelletted by centrifugation for 5 min at 12,000 rpm. The DNA pellet was washed with 70% cold ethanol, air dried and resuspended in 50 l of TE buffer (10 mm Tris-HCl, 0.1 mm EDTA, ph 8.0). RNA was degraded by treatment with RNase (50 g/ml) for 30 min at 37 ºC (Lee et al., 2000). DNA concentration and purity were measured using a spectrophotometer at 260 nm and 280 nm. RAPD primer and ampli cation condition For the development of the RAPD method for Fusarium isolates, the 4 different 10-mer primers (Kit A, Roth Germany) (Table 1) were screened for the amplification of template DNA

5 Morphological and molecular characterization o Fusarium spp. 29 from 30 isolates of Fusarium spp. Ampli cation reactions were performed in 25 l volumes containing 10 mm Tris- HCl (ph 9.0), 50 mm KCl, 0.1% TritonX-100, 1.5 mm MgCl 2, 0.1 mm of each datp, dctp, dgtp and dttp, 2 M of primer, 0.2 units of Taq polymerase, and approximately 100 g of genomic DNA. Ampli cations were performed in a Gradient DNA Thermal Cycler programmed for the following parameter : 94 ºC for 1 min, followed by 40 cycles of 94 ºC for 1 min, 36 ºC for 1 min, 72 ºC for 2 min, and a nal incubation at 72 ºC for 2 min (Achenbach et al., 1997). Ampli cation products (6 l of a 25 l reaction) were electrophoresed in 2% agarose gels with TBE running buffer, stained with ethidium bromide and either scanned into a computer imaging le or photographed (Sambrook et al., 1989). The computer program NTSYS-PC (version 2.1) was used to analyze the relationship among the Fusarium spp. studied. Similarity matrix was generated using the program Qualitative. This matrix was subjected to the unweighted pair group method with arithmetical averages (UPGMA). Cluster analysis was performed on the similarity matrix with the SAHN program using UPGMA and dendrogram was produced with the TREE program. TABLE 1. Code and nucleotide sequence of primers used in the random ampli ed polymorphic DNA (RAPD) reaction and G + C content. Primer 5 -sequence-3 G + C (%) A02 A03 A07 A09 TGCCGAGCTG AGTCAGCCAC GAAACGGGTG GGGTAACGCC RESULTS Isolation and Identi cation of fungi One hundred and fty seven isolates of Fusarium, 121 from roots and 36 from soil, were obtained from mulberry plantations with and without showing root rot symptoms (Table 2). Fusarium spp. were grouped and identi ed into 11 species. They included 102 isolates of F. solani, 26 of F. moniliforme, 16 of F. oxysporum, 3 each of F. phaseoli and F. dlamini, 2 of F. culmorum, one each of F. dimerum, F. graminearum,

6 30 Waraporn Sutthisa et al. F. beomiforme, F. scirpi and F. anthophilum. F. solani was signi cantly appeared to associate with all specimens investigated in this trial. TABLE 2. Frequency of isolation of fungi from roots and soils of mulberry plants exhibiting symptom of root rot and symptomless. Number of isolates Fungus Roots Soils Infected Non-infected Infected Non-infected F. solani F. oxysporum F. moniliforme F. phaseoli F. culmorum F. dlamini F. dimerum F. graminearum F. beomiforme F. scirpi F. anthophilum Total Detailed morphology of Fusarium species F. solani (Mart.) Appel & Wollenw. Macroconidia were type C, straight with blunt basal and apical cells. Microconidia were ellipsoidal on long simple phialides produced from the aerial mycelium. Chlamydospores appeared singly or in pairs (Figs. 1 5). F. oxysporum Schlecht. Macroconidia were type C, straight. Microconidia were comma shaped or ellipsoidal produced from short phialides in the aerial mycelium. Chlamydospores produced singly or in pairs (Figs. 1 5). F. moniliforme Sheldon Macroconidia were usually type B, narrow and straight, and sparsely produced. Microconidia produced in chains from simple phialides in the aerial mycelium (Figs. 1 5).

7 Morphological and molecular characterization o Fusarium spp. 31 F. phaseoli (Burkh.) T. Aoki & O Donnell Macroconidia were falcate and formed on slender conidiophores arising from hyphae. Microconidia with ellipsoidal, obovate or naviculate produced from slender aerial conidiophores on aerial mycelium. Chlamydospores formed frequently in mycelium and in conidia, mostly intercalary, single but sometimes in chains (Figs. 1 4). F. culmorum (W.G. Smith) Sacc. Macroconidia were very uniform in shape and produced from simple phialides with loosely branched conidiophores. Microconidia were absent. Chlamydospores appeared oval and formed singly or in chains (Figs. 1 2, 4 5). F. dlamini Marasas, Nelson & Toussoun Macroconidia were falcate and microconidia were ellipsoidal (Figs. 2 5). F. graminearum Schwabe Macroconidia were falcate produced from simple lateral phialides. Chlamydospores were absent (Figs. 1 2 and 5). F. beomiforme Nelson & Toussoun Microconidia, round or ellipsoid shaped were produced from simple phialides in the aerial mycelium (Figs. 1, 3 and 5). F. scirpi Lambotte & Fautr. Macroconidia had various sizes with elongated apical cells and produced from single or grouped phialides. Chlamydospores were globose and appeared in chains or clumps (Figs. 1 2 and 5). F. anthophilum (A. Braun) Wollenw. Microconidia were fusoid to allantoids and formed from simple phialides or complex phialides. Chlamydospore was absent (Figs. 1, 3 and 5). F. dimerum Penzig Only microconidia and chlamydospores were produced. O-shaped microconidia were produced on complex phialides. Chlamydospores produced intercalary and formed singly or in chains (Figs. 1, 3 and 5).

8 32 Waraporn Sutthisa et al. FIGURE 1. Colony morphology of Fusarium species on potato dextrose agar. A. F. solani; B. F. oxysporum; C. F. moniliforme; D. F. phaseoli; E. F. culmorum; F. F. dimerum; G. F. graminearum; H. F. beomiforme; I. F. scirpi; J. F. anthophilum. FIGURE 2. Macroconidia of Fusarium species. A. F. solani; B. F. oxysporum; C. F. moniliforme; D. F. phaseoli; E. F. culmorum; F. F. dlamini; G, F. graminearum; H. F. scirpi. Scale bar = 20 m.

9 Morphological and molecular characterization o Fusarium spp. 33 FIGURE 3. Macroconidia of Fusarium species. A. F. solani; B. F. oxysporum; C. F. moniliforme; D. F. phaseoli; E. F. dlamini; F. F. dimerum; G. F. beomiforme; H. F. anthophilum. Scale bar = 20 m. FIGURE 4. Chlamydospore of Fusarium species. A. F. solani; B. F. oxysporum; C. F. moniliforme; D. F. phaseoli; E. F. culmorumi; F. F. dlamini. Scale bar = 20 m. FIGURE 5. Conidiogenous cells of Fusarium species. A. F. solani; B. F. oxysporum; C. F. moniliforme; D. F. culmorum; E. F. dlamini; F. F. dimerum; G. F. graminearum; H. F. beomiforme; I. F. scirpi; J. F. anthophilum. Scale bar = 20 m.

10 34 Waraporn Sutthisa et al. RAPD analysis To investigate the degree of genetic diversity among and within collections of Fusarium spp., a total of 30 isolates comprising 12 of F. solani, 3 each of F. oxysporum, F. moniliforme and F. dlamini, 2 each of F. phaseoli and F. culmorum, 1 each of F. graminearum, F. beomiforme, F. scirpi, F. dimerum and F. anthophilum were analyzed, and RAPD pattern ampli ed with primer A02, A03, A07 and A09 (Fig. 6), to clearly differentiate between species. FIGURE 6. RAPD pro les of 30 isolates of Fusarium species obtained with primer A02, A03, A07 and A09. Lane M: Hyper ladder II molecular weight markers, Lane C: Control, Lanes 1 12: F. solani, lanes 13 15: F. oxysporum, lanes 16 17: F. phaseoli, Lanes 18 19: F. culmorum, lanes 20 22: F. moniliforme, lanes 23 25: F. dlamini, lane 26: F. graminearum, lane 27: F. beomiforme, lane 28: F. scirpi, lane 29: F.dimerum, lane 30: F. anthophilum.

11 Morphological and molecular characterization o Fusarium spp. 35 The PCR bands were more polymorphic between the species and less variable within the species. The high level of genetic variability among species of Fusarium allowed ngerprinting of most of the isolates by examining patterns produced by several primers. Twenty-four bands produced by 4 primers that showed polymorphism were scored. A phylogenetic tree was constructed (Fig. 7). The resulting dendrogram showed relations and genetic distances among different species. Genetic similarity coef cients ranged from 0.60 to 1.0 (Fig. 7). Two different major groups were obtained among the isolates. The rst group consisted of 20 isolates, which included 3 sub-groups (Ia, Ib, Ic). Sub-group Ia consisted of 12 isolates of F. solani, all of these isolates showed 100% genetic similarity. Subgroup Ib consisted of 7 isolates including F. dlamini, KKU03-2-1, KW04-2-2, F. dimerum, F. beomiforme, F. scirpi and F. anthophilum. Sub-group Ic consisted of 1 isolate of F. graminearum. A second I a I I b I c II a II II b Coef cient FIGURE 7. Dendrogram obtained from 30 isolates of Fusarium spp. with UPGMA. Isolates of different species are indicated at the termini of branches. The line below the dendrogram represents the similarity index.

12 36 Waraporn Sutthisa et al. group consisted of 10 isolates with two sub-group IIa and IIb. Sub-group IIa consisted of 3 isolates of F. oxysporum. Sub-group IIb consisted of 7 isolates including 2 isolates each of F. phaseoli, and F.culmorum and 3 isolates of F. moniliforme DISCUSSION The genus Fusarium contains many species of fungi that are commonly found in soil and on organic substrata and is widely distributed throughout the world (Burgess, 1981). Some species are capable of causing wilts, crown rots, root rots, or fruit rots. Others are opportunists because they colonize plant tissue after some type of stress debilitates the plant. Interestingly, many species of Fusarium that abound in the soil are not capable of causing disease. In conclusion, 11 species were isolated from root and soil around rhizosphere from mulberry trees. Base on morphological characteristics, the Fusarium spp. were identified as F. solani, F. oxysporum, F. moniliforme, F. phaseoli, F. culmorum, F. dlamini, F. dimerum, F. graminearum, F. beomiforme, F. scirpi and F. anthophilum. Consistly, Rodrigues and Menezes (2005) isolated and identi ed Fusarium spp. that obtained from seeds of cowpea by means of blotter tests and slide cultures. Species were differentiated according to the morphology of the macroconidia, microconidia and their arrangement in chains or false heads, the size and type of conidiophore, and the presence or absence of chlamydospores. The species were identi ed as F. semitectum, F. equiseti, F. oxysporum, F. solani, F. anthophilum, F. sporotrichioides, F. moniliforme and Fusarium sp. Latiffah et al. (2009) isolated three Fusarium species, F. oxysporum, F. proliferatum and F. solani, from roots and stem rots of Dendrobium orchids. Molecular characterization using PCR-RFLP of ITS + 5.8S regions showed that the isolates from the same species produced similar patterns and UPGMA analysis of PCR-RFLP of ITS + 5.8S clearly grouped F. oxysporum, F. proliferatum and F. solani into a separate cluster. RAPD-PCR analysis offers a convenient tool for characterization and analyzing variations of Fusarium spp. RAPD analysis has been applied widely in the detection and genetic characterization of phytopathogenic fungi (Lanfranco et al., 1995) including Khalil et al. (2003) who used RAPD analysis to study the taxonomic kinships among 5 Fusarium spp. Of 10 primers tested, four primers produced polymorphic amplification patterns with taxon speci c bands, in addition

13 Morphological and molecular characterization o Fusarium spp. 37 to individual speci c bands. Genetic analysis indicated 2 main clusters, with the minor cluster including all F. moniliforme and F. solani at the genetic similarity of GS = 57.82%. The major cluster consisted of all F. oxysporum, F. avenaceum and F. chlamydosporum clustered at 71% similarity. Different workers (Grajal-Martin et al., 1993; Achenbach et al., 1997; Nelson et al., 1997; Hyun & Clark, 1998; Migheli et al., 1998; Jana et al., 2003; Gupta et al., 2009) have grouped Fusarium spp. populations from different plant hosts using RAPD analysis and they suggested that RAPD markers can be a quick and reliable alternative for differentiating isolates of Fusarium spp. into their respective pathogenicity group. The polymorphisms observed for RAPD markers revealed a high degree of genetic diversity in Fusarium spp. at the inter-speci c level. Our study showed that RAPD technique is an in uential tool for discrimination different Fusarium species as well as Fusarium isolates within species. ACKNOWLEDGEMENTS This research is partially supported by the Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development Of ce (PERDO), Commission on Higher Education, Ministry of Education and Agricultural Biotechnology Research Center for Sustainable Economy, Khon Kaen University. REFERENCES Achenbach, L.A., Patrick, J.A. & Gray, L.E Use of RAPD markers as a diagnostic tool for the identi cation of Fusarium solani isolates that cause soybean sudden death syndrome. Plant Disease 80: Achenbach, L.A., Patrick, J.A. & Gray, L.E Genetic homogeneity among isolates of Fusarium solani that cause soybean sudden death syndrome. Theoretical Applied Genetics 95: Aoki, T., O Donnell, K., Homma, Y. & Lattanzi, A.R Sudden-death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex F. virguliforme in North America and F. Tucumaniae in South America. Mycologia 95: Booth, C Fusarium. Commonwealth Mycology Institute, Kew, Surrey, England. Burgess, L.W General ecology of the Fusaria. In: Fusarium disease, biology, and taxonomy. P.E. Nelson, T.A. Toussoun & R.J. Cook (Eds.). pp Pennsylvania University Press. Pennsylvania. Chancharean, S Sericulture technology. Sericulture Institute, Department of Agriculture, Ministry of Agriculture and Cooperatives. (in Thai). Chuprayoon, S., Chuprayoon, S., Sukorntasing P. & Lepayakun, P Mulberry and their control. Department of Agriculture,

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